CN102002086A - Method for preparing compound oleanolic acid from Xuanzhou pawpaw by cell culture method - Google Patents
Method for preparing compound oleanolic acid from Xuanzhou pawpaw by cell culture method Download PDFInfo
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- CN102002086A CN102002086A CN 201010285145 CN201010285145A CN102002086A CN 102002086 A CN102002086 A CN 102002086A CN 201010285145 CN201010285145 CN 201010285145 CN 201010285145 A CN201010285145 A CN 201010285145A CN 102002086 A CN102002086 A CN 102002086A
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- culture
- callus
- oleanolic acid
- cell
- fructus chaenomelis
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- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 title claims abstract description 37
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 title claims abstract description 37
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 229940100243 oleanolic acid Drugs 0.000 title claims abstract description 37
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 title claims abstract description 37
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 title claims abstract description 36
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing compound oleanolic acid from Xuanzhou pawpaw by a cell culture method, which comprises the following steps of: firstly, inoculating an MS solid culture medium with stems of Xuanzhou pawpaw as an explant, and inducing callus under the condition of 25+/-1 DEG C; then, optimizing culture through the subculture of the induced callus so that the callus achieves the loosen and luxuriant state and is applicable to suspension culture; then, inoculating the optimized callus in an MS liquid culture medium for suspension culture, optimizing again for luxuriant growth, and separating and purifying to obtain oleanolic acid under the culture condition of high secondary metabolism substances. The content of oleanolic acid in the culture achieves 6.88% and is higher than that in ripe Xuanzhou pawpaw fruits. The method of the invention has the characteristics of short culture period, high efficiency, no pollution to environment and the like.
Description
Technical field
The present invention relates to the method that a kind of cell suspension culture legal system is equipped with bio-pharmaceutical, specifically a kind of explant induction callus that utilizes Fructus Chaenomelis, and carry out cell suspension culture and produce triterpene compound---the method for Oleanolic Acid.
Background technology
Human utilization to the Secondary Metabolism of Plant material has long history, secondary metabolite is the important effective constituent of medicinal plant, but it is subjected to the restriction of plant biomass and secondary pollutant content, and some secondary substance is very little at natural content, thereby has limited to the widespread use of Secondary Metabolism of Plant material; Wild resource reduces and the Cultivar quality deterioration day by day simultaneously, brings many puzzlements for clinical use and quality control.Along with the rapid progress of biotechnology aspect, people accumulate in sight in the research that utilizes biotechnology to obtain plant secondary substance.For this reason, the scientists of correlative study is cultivated by the scale of vegetable cell through the exploration discovery of nearly decades, can realize Secondary Metabolism of Plant material " batch production " High-efficient Production.Utilize cell cultures production effective constituent, can alleviate resources of medicinal plant pressure,, poor growth strict for those growth conditionss, output is little, collection is difficult, be worth valuable plant amedica, has more significance in this way.
Produce secondary metabolites by the cell cultures of medicinal plant following advantage is arranged: (1) four seasons all can produce, and are not subjected to the influence of area, season and harmful organism.(2) floor space is few, but cell growth controls automatically and metabolic process is rationally regulated, and can carry out under artificial condition fully, can get rid of the puzzlement of disease and pest and pesticide residue, and quality that can strict control medicinal material is convenient to carry out large-scale industrial production.(3) be convenient to screen high yielding cell sarain.By optimizing the method for nutrient solution, culture condition and the good clone of selection, can obtain the secondary metabolite that content is higher than whole strain plant growing.Research data shows, has that kind of compound content in the histocyte of cultivating is higher than complete plant level surplus in the of 40.As the content of ginsenoside in the ginseng-cell of cultivating 5.7 times of natural phant; The content of Triptolide is 49 times of former plant content in the trypterygine culturing cell.(4) be beneficial to bio-transformation, seek new active drug composition.There are plurality of enzymes such as hydroxylase, oxydase, reductase enzyme, methylase, esterification enzyme, glycosyltransferase, Glycosylase in the vegetable cell, plant culture transforms xenobiontics as a kind of bio-reactor, it is unexistent to produce former plant, or even the still undiscovered compound of nature so far.(5) individual difference is little, and is with short production cycle, and equipment is simple, can save human and material resources etc.Can the people for conditions such as the certain temperature that provides, light application time, humidity, trophic hormones under the science of carrying out cultivate and produce.As required, set up different culture condition, breed growth in a large number, thereby can provide a large amount of virus-free seedling of high-quality and high yielding cell sarains to help automatization, large-scale production, enhance productivity by geometricprogression by different medicinal plant explants.(6) utilize bud mutation or the induced mutations that occurs in the tissue culture procedures, or carry out detoxification, cultivate new variety, improve the medicinal plant quality.(7) preserve germ plasm resource.
Fructus Chaenomelis has another name called cockle pawpaw, and formal name used at school is chaenomeles lagenaria Chaenomeles speciosa (Sweet) Nakai, belongs to Rosaceae Chaenomeles, the broad leaved and deciduous broad leaved shrub, and basic former for conventional Chinese medicine timber melon has very high pharmaceutical use and edibleness.The traditional Chinese medical science is thought and the effect that Fructus Chaenomelis has Shujin, active, spleen benefiting and stimulating the appetite, liver metastasis and acesodyne, dispels rheumatism be can be used for prevention and diseases such as treatment rheumatosis, cholera, dysentery, enteritis, vitamin B1 deficiency and vitamin C deficiency clinically.Oleanolic Acid is one of main pharmacodynamics composition of Fructus Chaenomelis.It is the five rings diterpene-kind compound, and molecular formula is C
30H
48O
3, be the main pharmacodynamics composition of pawpaw, pure product Oleanolic Acid is a white, needle-shaped crystals, fusing point is 308～310 ℃, and is water insoluble, dissolves in methyl alcohol, ethanol, ether, acetone and chloroform.Pertinent data shows that Oleanolic Acid exists with monomeric form in the pawpaw.
The major physiological function of Oleanolic Acid has:
1 antitumor action in recent years, people are mainly undertaken by following several respects OA antineoplastic action Mechanism Study: (1) Oleanolic Acid can anti-dna mutation, suppress the startup of canceration.(2) Oleanolic Acid has cancer eliminating effect and anti-invasion.(3) inducing apoptosis of tumour cell.(4) suppressing tumor vessel forms.In a word, the antitumous effect of OA has almost run through each stage of tumor development.
2 hepatoprotective effect Oleanolic Acid chemical structures have a plurality of active functional groups such as two keys, hydroxyl, carboxyl, and some chemical reactions easily take place, and as combining with the toxicity in vivo material, play and separate liver toxicity.The Oleanolic Acid pre-treatment also can prevent the emptying of the liver gsh of tetrachloro-methane induction.Experimentation on animals confirms that also metallothionein(MT) (MT) has increased nearly 30 times in pre-treatment can be with liver.Through clinical verification, Oleanolic Acid can effectively be treated viral hepatitis, is the main active ingredient of antiviral hepatitis medicine.
3 reducing blood-fat, hypoglycemic early stage bibliographical information, Oleanolic Acid can reduce the large and small mouse glucose level of artificial diabetes, and experimental hyperlipidemia rat and rabbit are all had tangible effect for reducing fat.
Effect is the new discovery of its pharmacological research to 4 pairs of cardiovascular effect Oleanolic Acids to cardiovascular disorder.There is research to point out that Oleanolic Acid does not have direct hypotensive effect to the experimental hypertension rat, but direct cardiotonic is arranged.When having step-down, cardiac stimulant and antiarrhythmic effect simultaneously, will be a fine selection for hypertensive patients stenocardia and patient in heart failure in view of it.
More to the mechanism of action and the action effect achievement in research of Fructus Chaenomelis Oleanolic Acid both at home and abroad at present.In pharmaceutically widespread use, aspect development of functional food, more and more come into one's own.The pawpaw dietotherapeutic, the existing fairly large Fructus Chaenomelis fruit that utilizes is processed can, preserved fruit, jam, fruit wine, fruit juice etc., and the kind that is made into protective foods is also more and more.The Fructus Chaenomelis nature and flavor are sweet flat to be slightly cold, nontoxic, edibility, and good for health.So existing market increases rapidly Fructus Chaenomelis and medicinal ingredients demand thereof, and the present mode of production can not satisfy the demand in market.
Summary of the invention
The invention discloses and a kind ofly prepare the method for compound Oleanolic Acid with the Fructus Chaenomelis cell culture method, technical problem to be solved be the explant that utilizes Fructus Chaenomelis to callus induce, the suspension culture of enlarged culturing, cell large scale produces Oleanolic Acid at last.
Technical scheme of the present invention is as follows:
A kind of method for preparing the compound Oleanolic Acid with the Fructus Chaenomelis cell culture method, with Fructus Chaenomelis stem section is starting raw material, comprise separating and extraction of the inducing of Fructus Chaenomelis callus, callus subculture optimization cultivation and cell suspension culture and Oleanolic Acid, may further comprise the steps:
(1) culture medium preparation
Preparation MS solid medium, every liter of distilled water adds 6-8g agar, 30-32g sucrose, 1mg2,4-D, 0.5mg KT regulates medium pH between 5.5-6.0.Be sub-packed in the triangular flask of 100ml, about every bottle of 25ml, carry out autoclave sterilization then, pressure is 1.0-1.5Kg/cm
2Between, the time is 20 minutes.
(2) be material with Fructus Chaenomelis stem section, alcohol-pickled 4-6s with 75%, be transferred to subsequently and carry out surface sterilization 6-10min in 0.1% mercuric chloride solution, the stem section is cut into the 0.8-1.2cm segment, be inoculated on the MS solid medium of having prepared, culture temperature is 25 ± 1 ℃, through the cultivation about 15～20d, obtains more callus in incision;
(3) callus that induces is separated from parent, it is carried out succeeding transfer culture, 25 ± 1 ℃ of temperature, be optimized by the cultivation to callus such as adjusting different sugar concentration, different pH value, different culture condition, the cycle of each regulation and control is about three weeks, behind the subculture, obtaining color is lurid loose callus more than 5 times, and the callus of this moment can be set up the suspension cell system;
(4) cell suspension culture
The loose callus of Fructus Chaenomelis with gained behind the subculture repeatedly is a test materials, is inoculated in the MS liquid nutrient medium and carries out suspension culture, and liquid nutrient medium does not add agar, and all the other are identical with the solid medium composition.Be sub-packed in the 250ml triangular flask, every bottled liquid measure is 95-100ml, and inoculum size is 3-5g, puts into homothermic suspension vibrator behind the access culturing bottle, and culture condition is: 25 ± 1 ℃ of temperature, shaking speed 110-120 rev/min, cultivated 20-22 days; By regulating different carbon sources, different initial pH, different hormones etc. are to the condition of suspension culture screening and optimizing.
(5) separation and Extraction Oleanolic Acid
The cell suspending liquid that obtains after suspending carries out suction filtration to it, collects the fresh callus above the filter paper, places 60 ℃ of oven for drying to constant-qualities to grind.Take by weighing callus powder 1g and place triangular flask, add 95% ethanol 25ml, mix, under ultrasound condition, extract 30min, distillating recovering solvent, the material water of the medicinal extract shape that obtains and chloroform washing are several times, merge washings, abandon water layer, the distillation chloroform, the acquisition Oleanolic Acid is with anhydrous alcohol solution and be settled to 25ml.Then it is carried out assay determination with ultraviolet spectrophotometer.
The present invention is a starting materials with Fructus Chaenomelis stem section, and explant is carried out inducing culture, sets up and filter out the suspension culture system that is fit to produce by cell culture method Oleanolic Acid.Design corresponding growth, produced culture medium prescription.This method has characteristics such as culture cycle is short, efficient is high, environmentally safe.
The present invention compares than prior art has following advantage:
(1) production process is controlled fully, is not subjected to the influence of physical environment;
(2) no agricultural chemicals and heavy-metal residual;
(3) extraction process is simple;
(4) low cost is saved in a large number and is ploughed, and the mixed economy that improves resource utilization and pomegranate economic forest is worth;
(5) production efficiency height: the high-content of the Oleanolic Acid of Fructus Chaenomelis cell suspension culture reaches 6.88%, far above the content of Oleanolic Acid in the Fructus Chaenomelis mature fruit.
Embodiment
Be example with the laboratory culture now, non-limiting examples is described below:
Be described in detail the technical process (being not limited to present embodiment) that Fructus Chaenomelis cell cultures of the present invention is produced the Oleanolic Acid compound with the embodiment form below:
1, culture medium preparation
Preparation MS solid medium, every liter adds 6-8g agar, 30-32g sucrose, 1mg2,4-D, 0.5mgKT regulates medium pH between 5.5-6.0.Be sub-packed in the triangular flask of 100ml, about every bottle of 25ml, carry out autoclave sterilization then, pressure is 1.0-1.5Kg/cm
2Between, the time is 20 minutes.
2, callus induces
With Fructus Chaenomelis stem section is material, alcohol-pickled 4-6s with 75%, be transferred to subsequently and carry out surface sterilization 6-10min in 0.1% mercuric chloride solution, the stem section is cut into the 0.8-1.2cm segment, be inoculated in the MS solid medium, culture temperature is 25 ± 1 ℃, through the cultivation about 15～20d, obtains more callus in incision;
3, the callus subculture is optimized
The substratum of configuration heterogeneity, with inductive callus subculture on different substratum, mainly be different nutrient media componentses, and different culture condition is continued to optimize the cell of subculture for obtaining loose vigorous suspension cell material the influence of callus growth.Each optimal conditions: 25 ± 1 ℃ of temperature, the low light level are according to cultivating about 20d.Pick out the color bright yellow, the loose vigorous callus of growth is carried out succeeding transfer culture, and the callus of optimization optimum regime carries out next step cell suspension culture.
4, cell suspension culture
With the loose callus of the Fructus Chaenomelis behind the subculture repeatedly is test materials, be inoculated in the MS liquid nutrient medium and carry out suspension culture, the composition of substratum (1) is identical, be sub-packed in the 250ml triangular flask, every bottled liquid measure is 95-100ml, and inoculum size is 3-5g, put into homothermic suspension vibrator after inserting culturing bottle, culture condition is: 25 ± 1 ℃ of temperature, shaking speed 110-120 rev/min, cultivated 20-22 days;
With the loose callus of the Fructus Chaenomelis behind the subculture repeatedly is test materials, be inoculated in and carry out suspension culture in the MS liquid nutrient medium, liquid nutrient medium does not add agar, all the other form identical with solid medium, be sub-packed in the 250ml triangular flask, every bottled liquid measure is 95-100ml, inoculum size is 3-5g, put into homothermic suspension vibrator after inserting culturing bottle. culture condition is: 25 ± 1 ℃ of temperature, shaking speed 110-120 rev/min, cultivate every bottle of bigger, maximum reached at 19.49g of cell fresh weight growth after 20 days, what culture Oleanolic Acid content was the highest reaches 6.88%, is higher than the total Oleanolic Acid content of Fructus Chaenomelis.Optimize culturing cell, make the eugonic while of cell can access the Oleanolic Acid of maximum production.
4, separation and Extraction Oleanolic Acid
The cell suspending liquid that obtains after suspending carries out suction filtration to it, collects the fresh callus above the filter paper, places 60 ℃ of oven for drying to constant-qualities to grind.Take by weighing callus powder 1g and place triangular flask, add 95% ethanol 25ml, mix, under ultrasound condition, extract 30min, distillating recovering solvent, the material water of the medicinal extract shape that obtains and chloroform washing are several times, merge washings, abandon water layer, the distillation chloroform, the acquisition Oleanolic Acid is with anhydrous alcohol solution and be settled to 25ml, then it is carried out assay determination with ultraviolet spectrophotometer.
Claims (1)
1. method for preparing the compound Oleanolic Acid with the Fructus Chaenomelis cell culture method, with Fructus Chaenomelis stem section is starting raw material, comprise separating and extraction of the inducing of Fructus Chaenomelis callus, callus subculture optimization cultivation and cell suspension culture and Oleanolic Acid, may further comprise the steps:
(1) culture medium preparation
Preparation MS solid medium, every liter of distilled water adds 6-8g agar, 30-32g sucrose, 1mg2,4-D, 0.5mg KT regulates medium pH between 5.5-6.0, is sub-packed in the triangular flask of 100ml, about every bottle of 25ml, carry out autoclave sterilization then, pressure is 1.0-1.5Kg/cm
2Between, the time is 18-22 minute;
(2) be material with Fructus Chaenomelis stem section, alcohol-pickled 4-6s with 75%, be transferred to subsequently and carry out surface sterilization 6-10min in the 0.1-0.12% mercuric chloride solution, the stem section is cut into the 0.8-1.2cm segment, be inoculated on the MS solid medium of having prepared, culture temperature is 24-26 ℃, through the cultivation about 15～20d, obtains more callus in incision;
(3) callus that induces is separated from parent, it is carried out succeeding transfer culture, temperature 24-26 ℃, be optimized by the cultivation to callus such as adjusting different sugar concentration, different pH value, different culture condition, the cycle of each regulation and control is about three weeks, behind 5-8 the subculture, obtaining color is lurid loose callus, and the callus of this moment can be set up the suspension cell system;
(4) cell suspension culture
The loose callus of Fructus Chaenomelis with gained behind the subculture repeatedly is a test materials, be inoculated in the MS liquid nutrient medium and carry out suspension culture, be sub-packed in the 250ml triangular flask, every bottled liquid measure is 95-100ml, inoculum size is 3-5g, puts into homothermic suspension vibrator behind the access culturing bottle, and culture condition is: temperature 24-26 ℃, shaking speed 110-120 rev/min, cultivated 20-22 days; By regulating different carbon sources, different initial pH values, different hormones etc. are to the condition of suspension culture screening and optimizing;
Wherein, the preparation of described MS liquid nutrient medium is identical with the described preparation of step (1) MS solid solid medium, does not just add agar;
(5) separation and Extraction Oleanolic Acid
The cell suspending liquid that obtains after suspending carries out suction filtration to it, collects the fresh callus above the filter paper, places 60 ℃ of oven for drying to constant-qualities to grind; Take by weighing callus powder 1g and place triangular flask, add 95% ethanol 25ml, mix, under ultrasound condition, extract 30min, distillating recovering solvent, the material water of the medicinal extract shape that obtains and chloroform washing are several times, merge washings, abandon water layer, the distillation chloroform, the acquisition Oleanolic Acid is with anhydrous alcohol solution and be settled to 25ml, then it is carried out assay determination with ultraviolet spectrophotometer.
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| CN101268758A (en) * | 2008-05-09 | 2008-09-24 | 南京林业大学 | Quick replication method for Xuan pawpaw tissue cultivation |
| CN101475929A (en) * | 2009-01-19 | 2009-07-08 | 东北林业大学 | Method for producing oleanolic acid by white birch suspension culture |
| CN101629161A (en) * | 2009-06-05 | 2010-01-20 | 东北林业大学 | Aralia elate seem hormone autotrophic cell line |
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| CN101475929A (en) * | 2009-01-19 | 2009-07-08 | 东北林业大学 | Method for producing oleanolic acid by white birch suspension culture |
| CN101629161A (en) * | 2009-06-05 | 2010-01-20 | 东北林业大学 | Aralia elate seem hormone autotrophic cell line |
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| 《河南中医学院学报》 20030731 张丽萍等 怀牛膝组织培养及齐墩果酸定量分析的研究 32-33 1 第18卷, 第107期 * |
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